Dihydroorotic acid



United States Patent DIHYDROOROTIC ACID Charles S. Miller, Lansdale, Edward L. Engelhardt, Ambler, and Janice T. Gordon, Ardmore, Pa., assignors to Merck & Co., Inc., Rahway, N. J., a corporation of New Jersey N Drawing. Application April 12, 1954, Serial No. 422,671

2 Claims. (Cl. 260-260) This invention relates to a new composition of matter, D-dihydroorotic acid, and more particularly to a compound which serves as an antimetabolite for nucleic acid synthesis. The compound of this invention has'the structural formula:

i IH 3=o 0:0 CH:

IH-CH-COOH Thisinvention also. comprises the process of synthesizing the D-, and L-, enantiomorphs as well as the DL- racemate of dihydroorotic acid from the corresponding D-, L-, and DL-N-carbethoxyasparagines. The products of the present invention are of interest because of their growth promoting or inhibiting properties with respect. to pyrimidine utilization in various strainsv ofv lactobacilli.

For some time chemists have been puzzled by the nature of the compound synthesized by M.'Bachtez and G. Cavallini, Ber. 66B, 681 (1933), which they called dihydroorotic acid. As discussed by Wright, L. D., Driscoll, C. A., Miller, C. S., and Skeggs, H. R., Proc. Soc. Exp. Biol. and Med, 84, 716 (1953), the addition of orotic acid to Lactobacz'llus bulgarz'cus 09 on a semi-synthetic medium results in good growth of the Lactobacillus bulgarz'cus O9. Ureidosuccinic acid in considerably larger amounts may be substituted for orotic acid. Both are D-, L-, or DL-carbethoxyasparag-ine NH: OOOH NHCH O 0 CH2 0 0 (3H I IHCHOOOH IQH-(il-COOH Ureidosuccinic acid Orotic acid i N NH: O 0 0H C O CH b'IH-(k-C O OH Ureidoiumaric acid Patented Dec. 11, 1956 The compound synthesizedby the method of Bachtez and Cavallini which they called dihydroorotic acid was tested for growth promoting activity with Lactobacillus bulgarz'cus O9 and was proved to be inactive. This was taken as evidence for the participation of ureidofumaric acid rather than dihydroorotic acid in the biosynthesis of orotic acid from ureidosuccinic acid. An enzyme system which reversibly converts DL-dihydroorotic to orotic acid but which would not utilize the dihydroorotic acid synthesized bythe method of Bachtez and Cavallini was recently discovered by J. Lieberman and A. Kornberg, Federation Proc. 12, 239 (1953). Unequivocal evidence de scribed by Miller, C. S., Gordon, J. T., and Engelhardt, E. L., J. Am. Chem. Soc. 75, 6086 (1953) shows that applicants synthetic procedure for the synthesis of DL dihydroorotic acid from N -carbethoxy-DL-asparagine yields a product which differs in melting point, infrared spectrum and solubility from the material prepared by the method of Bachtez and Cavallini. An attempt to resolve the material prepared by the method of Bachtez and Cavallini'into D and L isomers by recrystallization of its brucine salt was unsuccessful. There exists, then, as a result of the foregoing conclusive evidence that Bachtez and Cavallini did not in fact isolate dihydroorotic acid. As dihydroorotic acid is asymmetric about position 6, D and L forms as well as a DL racemate are theoretically possible.

The new compound D-dihydroorotic acid and the process for preparing L and DL dihydroorotic acid can be described as follows: D-, L-, or DL-, -carbethoxyasparagine is refluxed in an ethanol solution containing at least 2 molar equivalents of sodium ethoxide to give the sodium salt of dihydroorotic acid. Subsequent acidification gives the corresponding D-, L-, or DL-dihydroorotic acid in yields up to 70%.

Methyl alcohol has also been used in place of ethyl alcohol. Particular care must be exercised in acidifying the sodium salt of dihydroorotic acid to form dihydroorotic acid. The generation of heat during acidification may convert the dihydroorotic acid to ureidosuccinic acid. The process can be illustrated schematically as follows:

Sodium salt of D-, L-, or D-, L-, or DL-dihydroorotic DL-dihydroorotic acid acid A particularly novel feature of this invention is the fact that D-, L-, or DL-, N-carbethoxyasparagine respectively can be converted to the corresponding D-, L-, or DL-dihydroorotic acid. It is Well-known by experts in this art that synthetic procedures applied to asymmetric starting materials generally give the racemate product exclusively. Although it is true in this case that the asymmetric center of N -carbethoxyasparagine is not directly utilized during the course of reaction, one would expect racemization due to the presence of the alkaline environment. That this does not occur to any appreciable extent is evidenced by the small differential in optical rotation (00-275 obtained when dihydroorotic acid is converted to its brucine salt, recrystallized, and decomposed back to the free dihydroorotic acid. This small differential of (ad-2.75 represents the maximum amount of racemization obtained under reflux.

Microbiological tests also have demonstrated that L- dihydroorotic acid is utilized by some but not all of a number of pyrimidineless lactobacilli. When employed in large amounts D-dihydroorotic acid is an antimetabolite of L-dihydroorotic acid in pyrimidine utilization. The L- and DL-dihydroorotic acids are precursors tothe biological pyrimidines, i. e., thymine, uracil, cytosine and hydroxymethyl cytosine; these pyrimidines being basic structures of the nucleic acids. D-dihydroorotic acid has been strued as limiting the scope thereof.

Example I A solution of sodium ethylatewas prepared by dissolving (bright) (freshly cut) sodium (0.5 g., 0.02 mole) in dry ethanol (60 ml.) under reflux condenser and sodalime tube protection. To the warm solution was added N-carbethoxy-L-asparagine (2 g., 0.01 mole) and the mixture refluxed gently for four hours. Solid material begins separating immediately after the addition of the N-carbethoxy-L-asparagine. After cooling, the white solid was filtered, dried over P205 in vacuo and dissolved in a minimum of cold water. Immediately solution was made strongly acid to Congo red with concentrated hydrochloric acid while chilling. The resulting white solid was filtered and dried. Yield 1.11 g. (70%); dec. 266 C.; D+33.23 (C 1.992) in 1% NaHCOs.

Example 11 A solution of sodium ethylate was prepared by dissolving (bright) (freshly cut) sodium (0.5 g., 0.02 mole) in dry ethanol (60 ml.) under reflux condenser and sodalime tube protection. To the Warm solution was added N-carbethoxy-D-asparagine (2 g., 0.01 mole) and the mixture refluxed gently for four hours. begins separating immediately after the addition of the N-carbethoxy-D-asparagine. After cooling, the white solid was filtered, dried over PzOsin vacuo and dissolved in a minimum of cold water.

organisms which have a requirement mixture refluxed gently for four hours.

4 was made strongly acidic to Congo red with concentrated hydrochloric acid while chilling. The resulting white solid was filtered and dried. Yield 1.11 g. (70%); dec. 266

C.; (00 D-3l.54 (C 2.01) in 1% NaHCOa.

Example III A solution of sodium ethylate was prepared by dissolving (bright) (freshly cut) sodium (0.5 g., 0.02 mole) in dry ethanol ml.) under reflux condenser and sodalime tube protection. To the warm solution was added N-carbethoxy-DL-asparagine (2 g., 0.01 mole) and the Solid material begins separating immediately after the addition of the N-carbethoxy-DL-asparagine. After cooling, the white solid was filtered, dried over P20 in vacuo and dissolved in a minimum of cold water. Immediately the solution 7 was made strongly acidic to Congo red with concentrated Solid material Immediately the solution hydrochloric acid while chilling. The resulting white solid was filtered and dried. Yield 111 g. dec.

While the invention has been described with particular reference to specific embodiments, it is to be understood that it is not to be limited thereto but is to be construed broadly and restricted solely by the scope of the appended claims.

What is claimed is: I

1. D-dihydroorotic acid, said substance being in crystalline and substantially pure form and being substantially free from its optical antipode.

2. The method of preparing Ddihydroorotic acid that comprises heating N-carbethoxy-D-asparagine with at least 2 molar equivalents of sodium ethoxide, followed by acidifying the resultant solid and recovering the precipitated D-dihydroorotic acid product.

References Cited in the file of this patent Lieberman et al.: Federation Prac. 12, 239-240 (1953). 

1. D-DIHYDROOROTIC ACID, SAID SUBSTANCE BEING IN CRYSTALLINE AND SUBSTANTIALLY PURE FORM AND BEING SUBSTANTIALLY FREE FROM ITS OPTICAL ANTIPODE. 